T-cell Subset Enumeration Research Articles

Dietary therapy of murine primary biliary cholangitis induces hepatocellular steatosis: A cautionary tale.

There is increased interest in utilizing dietary interventions to alter the progression of autoimmune diseases. These efforts are driven by associations of gut microbiota/metabolites with levels of short-chain fatty acids (SCFAs). Propionate is a key SCFA that is commonly used as a food preservative and is endogenously generated by bacterial fermentation of non-digestible carbohydrates in the gut. A thesis has suggested that a diet rich in propionate and other SCFAs can successfully modulate autoimmunity. Herein, we investigated the effect of long-term administration of propionylated high-amylose resistant starches (HAMSP) on the course of murine primary biliary cholangitis. Groups of female ARE-Del mice were fed an HAMSP diet either before or after disease onset. A detailed immunobiological analysis was performed involving autoantibodies and rigorous T-cell phenotyping, including enumeration of T-cell subsets in the spleen, liver, intestinal intraepithelial lymphocytes and lamina propria by flow cytometry. Histopathological scores were used to assess the frequency and severity of liver inflammation and damage to hepatocytes and bile ducts. Our results demonstrate that a long-term propionate-yielding diet re-populated the T-cell pool with decreased naïve and central memory T-cell subsets and an increase in the effector memory T cells in mice. Similarly, long-term HAMSP intake reduced CD4+CD8+ double-positive T cells in intraepithelial lymphocytes and the intestinal lamina propria. Critically, HAMSP consumption led to moderate-to-severe hepatocellular steatosis in ARE-Del mice, independent of the stage of autoimmune cholangitis. Our data suggest that administration of HAMSP induces both regulatory and effector T cells. Furthermore, HAMSP administration resulted in hepatocellular steatosis. Given the interest in dietary modulation of autoimmunity and because propionate is widely used as a food preservative, these data have significant implications. This study also provides new insights into the immunological and pathological effects of chronic propionate exposure.

Live Rotavirus Vaccination Appears Low-risk in Infants Born to Mothers With Inflammatory Bowel Disease on Biologics

Biologic therapies in the context of inflammatory bowel disease and pregnancy lead to improved maternal and fetal outcomes. Placental transfer results in detectable drug concentrations in infants. Rotavirus infection results in diarrheal related hospitalizations; however, the live oral vaccine is not currently recommended in biologic exposed infants. The aim of this study was to assess the effect of maternal biologic therapies on the infant immune system and safety of live rotavirus vaccination in biologic-exposed infants. Biologic-exposed infants underwent standardized clinical assessments, drug concentration, and immune function testing (complete blood count, differential, immunoglobulin levels, extended B-cell and T-cell subset enumeration, Recent Thymic Emigrants, regulatory T-cell numbers, mitogen stimulation assays, and review of T-cell Receptor Excision Circles in the newborn screen). Rotavirus vaccine-specific adverse effects following immunizations up to 42 days post the last dose of the vaccine series were recorded. There were 57 infants born to 52 mothers with inflammatory bowel disease exposed to infliximab (n= 21), adalimumab (n= 19), vedolizumab (n= 10), and ustekinumab (n= 7) in the third trimester for a median of 39 weeks (interquartile range, 38-39 weeks) at delivery. Immunologic assessments validated for age were normal in all infants despite median infliximab concentrations of 6.1 ug/mL (range, 0.4-28.8 ug/mL), adalimumab concentrations of 1.7ug/mL (range, 0.7-7.9 ug/mL), ustekinumab concentrations of 0.6 ug/mL (range, 0-1.1), and undetectable for vedolizumab at 10.7 weeks (interquartile range, 9.4-12.4) of age. The live oral rotavirus vaccine series was provided to 50 infants with the first dose given at a median of 13weeks of age. No adverse effects following immunization were reported. Immune function testing was normal, and administration of live rotavirus vaccination appeared low-risk in biologic-exposed infants irrespective of circulating drug levels.

EuroFlow-Based Flowcytometric Diagnostic Screening and Classification of Primary Immunodeficiencies of the Lymphoid System.

Guidelines for screening for primary immunodeficiencies (PID) are well-defined and several consensus diagnostic strategies have been proposed. These consensus proposals have only partially been implemented due to lack of standardization in laboratory procedures, particularly in flow cytometry. The main objectives of the EuroFlow Consortium were to innovate and thoroughly standardize the flowcytometric techniques and strategies for reliable and reproducible diagnosis and classification of PID of the lymphoid system. The proposed EuroFlow antibody panels comprise one orientation tube and seven classification tubes and corresponding databases of normal and PID samples. The 8-color 12-antibody PID Orientation tube (PIDOT) aims at identification and enumeration of the main lymphocyte and leukocyte subsets; this includes naïve pre-germinal center (GC) and antigen-experienced post-GC memory B-cells and plasmablasts. The seven additional 8(-12)-color tubes can be used according to the EuroFlow PID algorithm in parallel or subsequently to the PIDOT for more detailed analysis of B-cell and T-cell subsets to further classify PID of the lymphoid system. The Pre-GC, Post-GC, and immunoglobulin heavy chain (IgH)-isotype B-cell tubes aim at identification and enumeration of B-cell subsets for evaluation of B-cell maturation blocks and specific defects in IgH-subclass production. The severe combined immunodeficiency (SCID) tube and T-cell memory/effector subset tube aim at identification and enumeration of T-cell subsets for assessment of T-cell defects, such as SCID. In case of suspicion of antibody deficiency, PIDOT is preferably directly combined with the IgH isotype tube(s) and in case of SCID suspicion (e.g., in newborn screening programs) the PIDOT is preferably directly combined with the SCID T-cell tube. The proposed ≥8-color antibody panels and corresponding reference databases combined with the EuroFlow PID algorithm are designed to provide fast, sensitive and cost-effective flowcytometric diagnosis of PID of the lymphoid system, easily applicable in multicenter diagnostic settings world-wide.

Diagnosis, immunological and post-mortem findings in two cases of human rabies virus encephalitis

Opportunistic viral infections are still a major complication following solid organ transplantation. Immune monitoring may allow the identification of patients at risk of infection and, eventually, the modulation of immunosuppressive strategies. Immune monitoring can be performed using virus-specific and non virus-specific assays. This article describes and summarizes the pros and cons of the different technical approaches. Among the assays based on non virus-specific antigens, the enumeration of T-cell subsets, the quantification of cytokines and chemokines and the quantification of intracellular adenosine triphosphate following mitogen stimulation are described and their clinical applications to determine the risk for viral infection are discussed. In addition, current specific methods available for monitoring viral-specific T-cell responses are summarized, such as peptide-MHC multimer staining, intracellular cytokine staining, enzyme-linked immunospot and virus-specific IFN-γ ELISA assays, and their clinical applications to determine the individual risk for opportunistic viral infections with human cytomegalovirus, Epstein-Barr virus and polyoma BK virus are discussed. The standardization of the procedure, the choice of the antigen(s) and the criteria to define cut-off values for positive responses are needed for some of these approaches before their implementation in the clinic. Nevertheless, immune monitoring combined with virological monitoring in transplant recipients is increasingly regarded as a helpful tool to identify patients at risk of infection as well as to assess treatment efficacy.

Immunologic Features of Cornelia de Lange Syndrome

Cornelia de Lange syndrome (CdLS) is a genetic syndrome with multisystem abnormalities. Infections are a significant cause of morbidity and mortality. The goals of our study were to identify the frequency and types of infections in CdLS and to determine if underlying immunodeficiency contributes to the clinical spectrum of this syndrome. We assessed infectious histories in 45 patients with CdLS and evaluated conventional immunologic screening tests in 27 patients. Among these 27 subjects, additional phenotypic enumeration of T-cell subsets, expression of activation markers in T cells, and production of cytokines in response to T-cell stimulants were studied in 12 CdLS subjects compared with 12 normal case control subjects. Recurrent infections were reported at high frequency in CdLS patients and included chronic ear infections (53%), chronic viral respiratory infections (46%), pneumonia (42%), sinus infections (33%), oral candidiasis (13%), sepsis (6%), and bacterial skin infections (4%). Full immune evaluation in 27 subjects led to identification of 9 cases of antibody deficiency syndrome in patients with severe forms of CdLS. Subjects with CdLS had decreased percentages of T regulatory cells and T follicular helper cells compared with normal control subjects (P < .05). This study identified for the first time a high frequency of antibody deficiency in CdLS subjects, indicating a critical need for screening and management of immunodeficiency in CdLS patients with a history of well-documented severe or recurrent infections. Furthermore, our results indicate that impaired T-cell populations may be associated with antibody deficiency in CdLS.

Evaluation of a dry format reagent alternative for CD4 T‐cell enumeration for the FACSCount system: A report on a Moroccan–Canadian study

Efforts to improve alternative CD4 T-cell counting methods are critical to accelerate the implementation of HIV antiretroviral therapy in resources limited regions. Substituting liquid format reagents to eliminate cold-chain transportation and refrigerated storage with dry format reagents contributes to higher efficiency supply management solution especially for laboratories at remote locations. ReaMetrix has developed dry format reagent kits compatible with the FACSCount system, a dedicated flow cytometer for T-cell subset enumeration widely used in resource limited settings. A dual site collaborative study was designed to compare T-cell subsets using both the new dry format ReaMetrix reagent and the original BD Biosciences liquid reagents. A total of 167 HIV positive samples prepared with Rea T Count (ReaMetrix) and FACSCount (BD Biosciences) reagents were analyzed using FACSCount Systems. To compare both methods, Bland-Altman, Pollock, Scott % similarity and correlation coefficient statistical analysis was applied. Immuno-Trol served as an assay processing control and quality indicator of interlaboratory and intralaboratory variation. The mean bias and limits of agreement for CD4 T-cell measurements between Rea T Count and FACSCount reagents were -16 cells/microl (-4.6%) and -74 to +43, respectively. The correlation obtained was 0.988 with a similarity of 97.9%. Between laboratory variation data was very good with %CV below 10%. The introduction of dry reagents permits the elimination of cold-chain transportation and the on-site refrigerated storage without compromise to assay quality. The substitution of dry reagents facilitates easier supply management practice that will assure wider access to quality HIV treatment.

Report of the Third Hungarian cell analysis conference: 16–18 May 2002, Budapest, Hungary

This conference was a successful continuation of previous conferences such as the Hungarian Cell Analysis Conference, Budapest, 1998 and 2000, and the ISAC-Sponsored International Conference for Flow Cytometry and Image Analysis, Epona, 1999. The Cell Analysis Section of the Hungarian Biophysical Society organized all four conferences. The popularity of the conference was hallmarked by the large number of participants (close to 300). The form of the conference proved to be very attractive: In the mornings, experts from specific fields delivered scientific lectures. In addition to Hungarian scientists, such as Margit Balazs, Gyula Hadlacky, Bela Molnar, Laszlo G. Puskas, Janos Szollosi, and Gyorgy Vereb, experts from abroad significantly raised the scientific standards of the conference. The international speakers included Peter Adorjan, Francis Mandy, Abe Schwartz, Howard M. Shapiro, and the authors of this introduction. In the afternoons, practical demonstrations were presented, ranging from basic techniques to stateof-the-art instrumentation. Practical training included cell culturing; mechanical cell separation for flow cytometry; methods for detection of cell proliferation; detection of apoptosis; fluorescence microscopy, image acquisition, and processing; confocal laser microscopy; fluorescence in situ hybridization; magnetic cell separation; magnetic mRNA isolation; real-time and traditional polymerase chain reaction; and detection of mutation. During the conference the attendees were able to choose six sessions of 11 topics. These demonstrations were headed by one of the speakers and sponsored by commercial companies that provided special kits and/or instruments to ensure a stable basis for successful practical training. An impressive selection of commercial companies provided contacts and improved awareness of advanced analytical cytology for the participants. The purpose of the conference was multifaceted. It served as a forum for presentations from Hungarian scientists, and, with the active participation of foreign experts, it opened a window to state-of-the art methods and techniques. The conference also facilitated the research cooperation between well-established scientists and Ph.D. graduate students, with the major purpose of the conference being education. The average age of the attendees was approximately 30 as more than 50% of the participants were Ph.D. students. Graduate students were able to earn credit points toward their final examination. The venue for the conference was the Medical University of Semmelweis in Budapest, the capital of Hungary. The university has great traditions, with its name reflecting the rich history of scholastic activity in Budapest. It was over 150 years ago that Ignaz Semmelweis completed his classic epidemiologic study in Budapest, studies that led to effective prophylaxis against childbed fever that still stands as the hallmark of evidence-based studies in epidemiology. At the conference both the lectures and the posters proved to be of high quality, demonstrating the high scientific standards of the community. The topics of the posters ranged from oncology (basic and applied research) to signal transduction, genetics, fertility research, and investigation in lower organisms such as Drosophila and earthworm. The methods applied were varied and highly sophisticated. Of 29 poster presentations, three prizes were awarded. Originally the poster awards committee (Margit Balazs and Attila Tarnok) was supposed to give only one award, but the decision was not easy because of the large number of excellent posters. With the financial help of commercial companies, the committee was able to present three poster awards instead of one. These awards were: First prize: Generation and Characterization by Flow Cytometry of Dendritic Cells, by Gizella Veszely, Janos Fent, Agnes Nagy, and Furesz Jozsef, Department of Pathophysiology, Institute for Health Protection of HDF, Budapest. Second prize: Hemocyte-Specific Molecular Markers in the Hematopoiesis and Innate Immunity of Drosophila melanogaster, by Istvan Nagy, Eva Kurucz, and Istvan Ando, Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, Szeged. Third prize: Astrocytes Support the Neuronal Differentiation of Neuroectodermal Progenitor Cells, by Vanda Szlavik, Zsuzsanna Kornyei, and Emilia Madarasz, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest. On the following pages, the reader will find the rich program of the Third Hungarian Cell Analysis Conference through the lecture and poster abstracts.

Reduction of variation in T-cell subset enumeration among 55 laboratories using single-platform, three or four-color flow cytometry based on CD45 and SSC-based gating of lymphocytes.

Enumeration of CD4(+) and CD8(+) T-cell subsets provides relevant information for diagnosis and monitoring of patients with cellular immunodeficiencies. As a result, an external quality assurance scheme was implemented in Belgium, The Netherlands, and Luxembourg in 1995. A workshop was held to train the participants in state-of-the art technology for assessment of absolute T-cell subset counts (i.e., a three or four-color, single-platform assay with lymphocyte gating based on CD45 and sideward light scatter) with the aim to achieve between-site coefficients of variation (CVs) <10% and within-site CVs <5% for > or =75% of the participants. Three send-outs of stabilized blood from a healthy donor were distributed to 55 laboratories, each with the request to perform the standard assay on three occasions. For comparison, each laboratory performed its local technique in parallel. With the standard technique, between-site CVs of approximately 8% (CD3+ T cells), approximately 9% (CD4+ T cells), and approximately 10% (CD8+ T cells) were achieved. Within-site CVs were <5% for 82% (CD3+ T cells) and approximately 70% (CD4+ and CD8+ subsets) of the participants. Local techniques yielded between-site CVs of 13%-17% for CD3+, CD4+, and CD8+ T cells. The state-of-the-art technology for T-cell subset enumeration was implemented successfully among 55 Belgian-Dutch laboratories and resulted in significant reductions of between-site variation of absolute CD3+, CD4+, and CD8+ T-cell counts.

T-cell subset counting and the fight against AIDS: reflections over a 20-year struggle.

The story of T-lymphocyte subset immunophenotyping technology is reviewed on the occasion of the 20th anniversary of CD4 T-cell enumeration. Over time, immunophenotyping has evolved into precise, reliable, but complicated and expensive technology requiring fresh blood samples. The gating technologies that were universally adapted for clinical flow cytometry for the past decade relied on rapidly deteriorating morphological scatter characteristics of leukocytes. This special issue dedicated to CD4 T-cell enumeration features most of the available new options that will have a significant impact on how this technology will be implemented within the first decade of the 21st century. In a series of original publications, including the new NIH guideline for T-cell subset enumeration, contemporary gating protocols that use immunologically logical parameters are presented as part of the more reliable and affordable immunophenotyping alternative. Some of the improvements addressed here include the costs of the assays and the capacity to monitor interlaboratory and intralaboratory performances. It is clear that an effective attack on the human immunodeficiency virus (HIV) epidemic has to embrace resource-poor regions. Reducing the cost of the assay while improving reliability and durability is a move in the right direction.

Selection of lymphocyte gating protocol has an impact on the level of reliability of T-cell subsets in aging specimens.

In the past decade, human immunodeficiency virus (HIV) lymphocyte immunophenotyping has evolved significantly. New fluorochromes, new multicolor reagents, enhanced instruments, and the capacity to provide absolute cell counts using the single-platform technique have all contributed to the reliability of T-cell subset measurements. In this study, four gating protocols were evaluated to select the most robust method for T-cell subset enumeration. Peripheral blood specimens from 21 HIV(+) and 20 HIV(-) individuals were monitored up to 96 h. Aliquots of specimens were stored at room temperature and analyzed at 6 (baseline), 48, 72, and 96 h. Aliquots were stained with CD45-fluorescein isothiocyanate (FITC)/CD3PC5/CD4RD1/CD8ECD. Data analysis was performed with all four gating protocols. Only with fresh blood did all protocols provide similar results. From samples that were 48 h old, the choice of gating strategy had a dramatic impact on immunophenotyping results. The largest deviations from baseline values occurred at 96 h and gating protocols that included dual light scatter gates provided the greatest shift of T-cell subset values over time. The gating protocols that were based exclusively on cell lineage-specific gates gave the most robust T-cell values up to 96 h. By selecting the appropriate gating protocol, the temporal integrity of specimens can be extended up to 4 days.

Impact of unified procedures as implemented in the Canadian Quality Assurance Program for T lymphocyte subset enumeration

The objective of the Canadian Quality Assurance Program (CQAP) is to provide the most reproducible and accurate T-cell subset enumeration for individuals living with HIV who are enrolled in the Canadian Clinical Trial Network for Human Immunodeficiency Virus (HIV) and Acquired Immune Deficiency Syndrome (AIDS) Therapies (abbreviated as CTN). The Canadian National Laboratory for Analytical Cytology, within the Laboratory Centre for Disease Control, is part of the Health Protection Branch of Health Canada. For the past eight years, the Laboratory for Analytical Cytology has been responsible for delivering a bilingual quality assurance program for CD4 T-cell enumeration. This federal program, which integrates biotechnology transfer with quality assessment, was achieved through the organization of workshops focused on technology transfer and essential skill-building techniques. Two training sessions were conducted for the CTN flow cytometer operators. The first introduced the concept of window of analysis, to demonstrate the practical benefits of unified quantitative fluorescent measurement. As a follow-up to the first workshop, participants performed a series of quantitative assays that monitored the expression of CD69, an early activation marker. This quantitative fluorescence protocol was performed with acceptable inter-laboratory variation using modified commercial kits. The second workshop focused on a absolute count method based on a single platform. Four preserved whole-blood preparations were tested with this approach. The combined effort reduced inter-laboratory variation. The direct impact was monitored as related to the frequency of participation. Over the years, the standard deviation of average accumulated variation decreased dramatically with increased frequency of participation, from 10% to <4%. Cytometry 33:146–155, 1998. © 1998 Wiley-Liss, Inc.

Impact of unified procedures as implemented in the Canadian Quality Assurance Program for T lymphocyte subset enumeration

The objective of the Canadian Quality Assurance Program (CQAP) is to provide the most reproducible and accurate T-cell subset enumeration for individuals living with HIV who are enrolled in the Canadian Clinical Trial Network for Human Immunodeficiency Virus (HIV) and Acquired Immune Deficiency Syndrome (AIDS) Therapies (abbreviated as CTN). The Canadian National Laboratory for Analytical Cytology, within the Laboratory Centre for Disease Control, is part of the Health Protection Branch of Health Canada. For the past eight years, the Laboratory for Analytical Cytology has been responsible for delivering a bilingual quality assurance program for CD4 T-cell enumeration. This federal program, which integrates biotechnology transfer with quality assessment, was achieved through the organization of workshops focused on technology transfer and essential skill-building techniques. Two training sessions were conducted for the CTN flow cytometer operators. The first introduced the concept of window of analysis, to demonstrate the practical benefits of unified quantitative fluorescent measurement. As a follow-up to the first workshop, participants performed a series of quantitative assays that monitored the expression of CD69, an early activation marker. This quantitative fluorescence protocol was performed with acceptable inter-laboratory variation using modified commercial kits. The second workshop focused on a absolute count method based on a single platform. Four preserved whole-blood preparations were tested with this approach. The combined effort reduced inter-laboratory variation. The direct impact was monitored as related to the frequency of participation. Over the years, the standard deviation of average accumulated variation decreased dramatically with increased frequency of participation, from 10% to <4%.

Effect of environmental enrichment and housing density on immune system reactivity to acute exercise stress

Positive stress has been described in the literature but not well characterized experimentally. This experiment was designed to test the hypothesis that environmental enrichment and housing density in C57BL/6 female mice modulate immune responses to acute exercise stress. A 2 × 2 × 2 factorial design was used where enrichment (enrich), nonenriched (nonenrich), group housed (group), individually housed (ind), and stress (stress) or no stress (nonstress) acted as the independent variables. Enrichment involved a 7-week exposure to in-cage running wheels and a variety of cage objects. Ninety minutes after treadmill exercise stress at 25 m/min, 4° slope, for 30 min (or no exercise stress) mice were sacrificed, and splenocyte blastogenesis to the T-cell mitogen concanavalin A (Con A), splenic T-cell subset enumeration, and percent live/dead splenic cells by flow cytometry were evaluated. Results showed significant interaction effects for mitogen responses, percent Ly2 + subset, and percent live/dead splenocyte responses. Proliferation to Con A was higher in nonenrich group-housed animals than other groups. Percent live/dead cell analysis revealed a significant housing × stress interaction with fewer percent live and higher percent metabolically stressed splenocytes obtained from ind-stressed mice than other groups. These data suggest that enrichment and housing density are important factors influencing immune responses in the basal state, and in response to exercise stress.

Development of AIDS in a cohort of HIV‐seropositive homosexual men in Australia

The Sydney AIDS Prospective Study is a cohort study of 1057 homosexual men enrolled between February 1984 and January 1985. By June 30, 1989, 111 (26.8%) of the 414 men who were seropositive for antibodies to human immunodeficiency virus (HIV) at enrollment had developed the acquired immunodeficiency syndrome (AIDS). On univariate analysis the following baseline factors were significantly associated with subsequent development of AIDS: splenomegaly; a lymphocyte count less than 1500 x 10(6)/L; a percentage of CD4+ cells less than 20% of the total lymphocyte count; an absolute number of CD4+ cells less than 200 x 10(6)/L; and a CD4+: CD8+ ratio less than 1.00. In a proportional hazards model the following factors retained significance: a lymphocyte count less than 1500 x 10(6)/L; an absolute number of CD4+ cells less than 200 x 10(6)/L; and a CD4+: CD8+ ratio less than 1.00. A CD4+ cell count less than 200 x 10(6)/L carried the greatest relative risk (3.99) for the development of AIDS. This study has confirmed that the appreciable rates of progression to AIDS demonstrated in overseas cohorts of HIV-infected persons also apply in the Australian context. A number of laboratory variables was found to be predictive for the subsequent development of AIDS. As we were not able to determine accurately whether subjects were receiving antiretroviral treatment or prophylaxis for opportunistic infections the observed rates in this study should be seen as minimum estimates. These findings have important implications for HIV-infected persons and for public health planning, and emphasise the need for regular clinical monitoring and T-cell subset enumeration in HIV-infected persons.

Effect of specific chemotherapy on the immune response and resistance to reinfection in experimental schistosomiasis mansoni

Mice infected for 45 days with 120 Schistosoma mansoni cercariae and treated with praziquantel in a dose of 500 mg/kg for two consecutive days had a significant lower resistance to reinfection when challenged two weeks after treatment (45% compared to 88% in infected challenged untreated mice). In praziquantel-treated mice, the reduction in the per cent resistance was accompanied by a diminution in the size of hepatic granulomata and its in vivo correlate the delayed foot pad swelling. Moreover, the granuloma proportionate T-cell subset enumeration revealed a significant reduction in the number of T-helper cells. The humoral immune response as measured by the immediate foot pad swelling was not affected by praziquantel. Results reveal besides the diminution of the state in resistance to reinfection after praziquantel, possible involvement of egg-related pathology as a T-cell mediated reaction and as a mechanical obstacle in maintenance of this resistance.

The Sydney AIDS Project: development of acquired immunodeficiency syndrome in a group of HIV seropositive homosexual men.

The Sydney AIDS Project is a prospective immunoepidemiological study of 996 homosexual/bisexual men enrolled between February 1984 and January 1985. By January 1987, 32 of 386 homosexual men who were seropositive at enrollment in the study had developed AIDS, yielding a crude progression rate of between 2.8% and 4.2% per annum. Of these subjects, 23 (72%) developed AIDS within 12 months of enrollment. In univariate analysis, the only lifestyle differences between seropositive subjects who progressed to AIDS and those that did not progress were less frequent oral sex activity and more use of marijuana in the three months prior to enrollment. In multivariate analysis, seropositive subjects who progressed to AIDS were more likely to have a lower percentage of CD4+ cells, a higher percentage of CD8+ cells and to have used marijuana in the three months prior to enrollment than the seropositive subjects who did not progress. No HIV seropositive subject who was asymptomatic and had normal T-cell subsets at enrollment had developed AIDS by January 1987. Persistent generalised lymphadenopathy was not associated with progression to AIDS. Although there are a number of lifestyle factors that may be associated with HIV infection, this study did not implicate most of these in the progression of HIV seropositive subjects to end-stage AIDS. We conclude that antecedent changes in T-cell subsets are associated with progression to AIDS and we emphasise the prognostic value of enumeration of T-cell subsets in HIV seropositive persons.

CONTRASTING RELATIONS BETWEEN SUPPRESSOR-CELL FUNCTION AND SUPPRESSOR-CELL NUMBER IN CHRONIC LIVER DISEASE

75 patients with various chronic liver diseases were investigated by simultaneous assay of suppressor-cell function and enumeration of suppressor T cells by monoclonal antibody. In chronic active hepatitis and primary biliary cirrhosis, substantial defects in suppressor-cell function were associated with negligible alterations in the proportions of suppressor cells. Defective suppressor-cell function was also evident in most patients with HBsAg-positive liver disease and in some with alcoholic liver disease. While the expected relations between suppressor-cell function and the proportions of helper or suppressor cells and the helper/suppressor ratio were apparent in alcoholic liver disease, the results in the HBsAg-positive, HBeAg-positive patients were in direct contrast. This apparent anomaly may reflect functional heterogeneity of the lymphocyte population detected by the monoclonal antibody OKT8, which identifies suppressor and cytotoxic cells. This study demonstrates that in the absence of simultaneous measurement of function, enumeration of T-cell subsets with monoclonal antibodies is an inadequate assessment of immunoregulatory balance.